The expression of polypeptides on the surface of bacteria and bacteriophages has been pursued for several years, in part because of interest in recombinant antibody production. Many other potential applications exist, including the production of genetically-engineered whole cell adsorbents, construction of “peptide libraries”, cell bound enzymes, and use as live vaccines or immunogens to generate antibodies.
In bacteria, one approach to obtaining surface expressed foreign proteins has been the use of native membrane proteins as a carrier for a foreign protein. In general, most attempts to develop methods of anchoring proteins on a bacterial surface have focused on fusion of the desired recombinant polypeptide to a native protein that is normally exposed on the cell's exterior with the hope that the resulting hybrid will also be localized on the surface.
In a prior invention (Norwegian patent application no. 20033176) the present inventors also provided an expression system where a heterologous polypeptide (termed “desired” protein) was expressed in the bacteria Methylococcus capsulatus. The heterologous protein is preferably linked to an outer membrane protein in M. capsulatus termed MopE.
MopE has a 540 amino acid protein sequence, with a short (29 amino acids) N-terminal sequence dependent signal sequence, followed by a N-terminal domain (176 amino acids) and a C-terminal domain (335 amino acids). The N-terminal domain is not secreted, while the C-terminal domain is secreted and expressed on the cell surface. The MopE amino acid sequence is shown in the sequence listing, as SEQ. ID. NO. 1
The method of secretion is not known. MopE does not show high sequence similarity to other known secreted proteins. The secretion is host specific, although inserted in the IM and released to the periplasm, the MopE protein is not secreted by E. coli hosts, only by M. capsulatus. MopE has been proposed to be secreted either by the Type 2 Secretion system (T2S) or the Type 5 Secretion system (T5S) based upon its primary sequence (Fjellbirkeland et al., 2001). Proteins secreted by the T2S or T5S systems can be translocated across the IM either by the Tat or the Sec machinery, which are shown in FIG. 1.
Secretion of T2S substrates is very host specific. Secretion by the T2S system (FIG. 1) is a two-step process in which the secreted protein initially is exported across the IM by the Sec or Tat export systems. The periplasmatic intermediate is subsequently translocated across the OM by the T2S secretion through the channel formed by the secretin that is large enough to translocate folded or close to folded substrates. T2S is energized by ATP, and although not required for secretion, the proton motive force increases the rate of secretion. Substrates of the T2S system share no obvious similarities in primary sequence. Although no recognition signal that confine proteins to secretion by the T2S pathway has been identified, a potential common feature for T2S substrates is a medium to high content of β-sheet (de Vries et al., 1990 and Sandkvist, 2001). β-strands were predicted in the N-terminal domain of MopE by the computer program PRED-TMBB (http://bioinfromatics.biol.uoa.gr/PRED-TMBB), and this program also predicted an N-terminal β-barrel in MopE. The predicted β-strands may form the tertiary structures of β-sheets required for translocation by the T2S pathway. In addition to the prediction of such structures in N-terminal domain of Mop-E, MopEC is heat-modifiable while MopE* is not (Fjellbirkeland et al., 2001), indicating that the N-terminal domain of MopE indeed contains stabile β-structures, and thus is a candidate T2S substrate. Since a Sec-compatible signal sequence has been predicted in MopE it has been considered likely that the Sec machinery export MopE across the IM.
However, the presence of β-structures does not confine secreted proteins to the T2S route. Substrates of T5S, autotransporters, require a β-domain that forms a β-barrel that allows for translocation of the passenger domain across the OM. Since the N-terminal domain of MopE has the potential of containing β-structures, the domain could function as an autotransporter translocation unit in T5S. No autoproteolytic activity could be demonstrated for MopE using the substrate azocasein, however, such activity is not a widely distributed feature among T5S substrates, and thus not required. And while autotransporters inherently contain all information and accessory factors necessary for translocation to the OM, release of the protein to the extracellular space most often require cleavage by an external protease. Thus secretion by T5S is somewhat dependent on the host cell, and the host specificity observed for the secretion of MopE does not exclude secretion by the T5S route.
Thus, it was thought that the most likely mechanism for translocation of MopE was either by the T2S or T5S routes, by the Sec or Tat transport systems. This translocation would then require the β-structures found in the N-terminal domain of MopE. Thus it was expected that the removal of the N-terminal domain of MopE would abolish the ability of the truncated protein, here termed MopEH*, to translocate. The MopEH* amino acid sequence is shown in the sequence listing, as SEQ. ID. NO. 2.